This invention relates generally to the chemical synthesis of certain novel nucleotide sequences and novel synthetic peptides and, more particularly, to their use in diagnostic tests for M. tuberculosis and their immunoprophylactic value.
Tuberculosis is a serious infectious disease which affects 30 million people worldwide, especially in the developing countries (World Health Organization, Bull. WHO, 61, 779, 1983).
The diagnosis of tuberculosis relies on the observation of acid-fast bacilli in clinical specimens and on PPD (Purified Protein Derivative), a delayed type cutaneous hypersensitivity test (DCH). However, very often the number of bacterial cells in the sample is insufficient to make a successful diagnosis of the disease. On the other hand, the utility of PPD is limited both by its lack of specificity and by its inability to distinguish between an active disease state, previous sensitization by contact with M. tuberculosis, or cross-sensitization to other mycobacteria. The use of peptides as tools in the diagnosis of mycobacterial diseases was discussed recently in the First Vaccilep Workshop on the Immunology of Leprosy. (Immunology today. 10: 218-221, 1989.) The application of this strategy to tuberculosis would enable the production of highly specific and very stable reagents, at low cost, which could be used in immunoassays of excellent reproducibility. This type of easy-to-perform test would be useful in both seroepidemiological and clinical studies, looking to tuberculosis control and prevention. Besides, much attention has been focused on the use of nucleic acid probes to specifically detect a mycobacterial infection.
BCG (Bacillus Calmette Guerin) has been the most widely used vaccine around the world. However, it has not been possible to clearly demonstrate its protective value in all the immunization trials carried out to date.
The knowledge of individual antigens of M. tuberculosis is very important in the search for immunoprophylactic molecules and in the detection of specific molecules, i.e., antigens, exclusively present in M. tuberculosis. Such type molecules could be used in the design of reagents to accurately diagnose a tuberculosis infection, both at the DNA and the protein level, thus circumventing the cross-reactivity problems associated with the current diagnostic tests, and also they could serve as potential vaccines against this threatening disease.
The application of recombinant DNA techniques to the study of M. tuberculosis genes, has provided the complete nucleotide sequences which encode proteins of 71, 65, 38, 32 and 19. Despite the fact that many of these genes encode for M. tuberculosis antigens which belong to the group of ubiquitous Heat Shock Proteins, immunological studies have demonstrated the presence of some epitopes of these molecules, most of which are capable of eliciting cellular responses "in vitro".